It is possible that a mucosal vaccine approach that could induce potent mucosal immunity at the point of initial infection in the lower respiratory mucosa could prevent TB infection. By preventing initial TB infection, latent TB infection with the risk for reactivation TB disease would be prevented. There have been no systematic studies of this vaccine concept in any animal model or in humans despite its potential for a huge impact on the overall prevalence of TB infection and disease. In this R21 application, we propose to use modem mucosal vaccination approaches to directly determine whether potent mucosal immune responses can protect against TB infection. Our project will test 2 major hypotheses: 1) Intranasal priming with recombinant mycobacterial proteins and boosting with BCG will induce optimal TB-specific Th1, CTL and secretory IgA responses in the lung. We will use a promising new recombinant TB vaccine candidate, Mtb72f, developed by Corixa Corp. in these studies, alone and in combination with BCG and/or different adjuvants. Dose optimization and prime/boosting experiments will be done giving combinations of recombinant protein, DNA vaccines and BCG intranasally. CpG + choleratoxin will be given as intranasal adjuvants for recombinant protein vaccinations. DNA vaccines will be given intranasally formulated in liposomal preparations. 1 month after the final booster vaccination, lung lymphocytes, spleen cells and bronchoalveolar lavage fluid will be harvested and studied for antigen specific IFN(, CTL and slgA/IgG responses. 2) Optimal Regional TB-specific Th1, CTL and secretory IgA responses in the lung can provide resistance against initial pulmonary TB infection. The systemic and mucosal prime/boosting strategies found to induce optimal Mtb72f- and BCG-specific immune responses in the experiments outlined above will be studied for the ability to protect against aerosol challenges with mycobacteria. Initially, we will perform aerosol challenges with recombinant BCG expressing a reporter gene (T. cruzi cruzipain vs GFP). In addition to CFU counts, more highly sensitive assays will be used to detect infection in the lungs post-challenge. Real-time PCR assays will be done to detect the presence of the T. cruzi cruzipain gene in lung and peripheral tissues post-challenge. Infection by GFP expressing BCG will be detected by fluorescent evaluation of tissue sections. Prime/boosting strategies found to prevent BCG infection after aerosol challenges will be studied for the ability to prevent infection with virulent M. tuberculosis by aerosol challenge.